EP0704534A2 - Vecteur ADN viral récombinant pour la transfection de cellules animales - Google Patents
Vecteur ADN viral récombinant pour la transfection de cellules animales Download PDFInfo
- Publication number
- EP0704534A2 EP0704534A2 EP95306498A EP95306498A EP0704534A2 EP 0704534 A2 EP0704534 A2 EP 0704534A2 EP 95306498 A EP95306498 A EP 95306498A EP 95306498 A EP95306498 A EP 95306498A EP 0704534 A2 EP0704534 A2 EP 0704534A2
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- EP
- European Patent Office
- Prior art keywords
- vector
- recombinase
- gene
- sequence
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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Definitions
- the present invention relates to a recombinant DNA viral vector for transfecting an animal cell. More particularly, the present invention relates to a recombinant DNA viral vector comprising a recombinase gene or a DNA sequence coding for a recombinase-recongnizing sequence, a method for transducing a foreign gene into an animal cell using said vector, and use thereof in gene therapy.
- Retrovirus has been often employed as a viral vector for gene transduction.
- retrovirus is transfected only into mitotic cells and integrated into a chromosome of host cells, and retrovirus as a viral vector, therefore, encounters a problem from a viewpoint of safety, especially in gene therapy. It is thus considered that retrovirus should be limitedly used as a viral vector.
- An adenoviral vector is advantageous in that it shows a transducing efficiency of almost 100% in a variety of animal cultured cells, has no positive mechanism for integration into chromosome unlike retrovirus, and can transduce a gene even into a resting cell.
- an adenoviral vector is considered as being applicable over an extremely wide fields for attempting to transduce a foreign gene. It would thus be established in the near future that an adenoviral vector is used as one of major technology for gene therapy.
- An adenovirus vector has been widely utilized as one technology for gene therapy or for researching an expression in highly differentiated cells such as a nervous system cell.
- an in vivo gene therapy has been extensively studied wherein a gene which is defective in a living cell is transduced into the cell by direct injection of the gene into a tissue in which the cell exist.
- five research groups have been already allowed to conduct a clinical trial for treating patients with cystic fibrosis by the in vivo gene therapy.
- researches with gene therapy have also been extended to muscular dystrophy, familial hypercholesterolemia, and brain tumor.
- an adenoviral vector enables transduction of a gene even into a resting cell. Therefore, an adenoviral vector has been utilized for transduction of a gene into differentiated cells, especially into a nervous system cell, when conducting experiments on gene transduction into a primary culture cell or animal body.
- an adenoviral vector will be into practice particularly in gene therapy, because the vector enables an expression of a gene by direct injection or administration into animal body, as well as transduction of a gene into various differentiated and non-differentiated cells including a nervous system cell.
- an adenoviral vector Unlike a retrovirus, an adenoviral vector, however, lacks any positive mechanism for integration into chromosome, resulting in that an expression of a gene in the vector occurs only temporarily. That is, the expression continues only for a few weeks, at most for about 2 months. Thus, when the therapeutic effect has to be maintained, the injection or administration of the vector should be repeated for the continuous expression. However, the repeated injections or administrations might induce the generation of an antibody reducing the therapeutic effect.
- an object of the present invention is to provide a recombinant adenoviral vector system wherein a foreign gene is transduced into an animal cell by an adenoviral vector and then converted into a form capable of autonomously replicating within the cell.
- a further object of the present invention is to provide such a system for in gene therapy.
- the present inventors have made extensive researches and succeeded in obtaining a recombinant adenoviral vector system: wherein an expression unit bearing a foreign gene is transduced into cells with an adenoviral vector, and then converted into a circular DNA molecule by the use of a recombinase gene and a recombinase-recongnizing sequence and, wherein a replication origin has been further introduced into the thus formed circular DNA molecule, whereby the gene expression unit bearing the foreign gene is capable of autonomously replicating to continue in expressing the foreign gene in the cells.
- a recombinase refers to a specific recombination DNA enzyme, which is capable of recognizing a specific DNA sequence composed of several tens of base pairs to cleave the sequence and changing DNA fragments formed from such cleavages therewith to religate those fragments to produce a new DNA sequence.
- both a recombinant adenoviral vector expressing the recombinase and a recombinant adenoviral vector having two copies of the recombinase-recongnizing sequence at the same orientation are constructed, and both two vectors are co-transfected into a cell, wherein the recombinase is expressed in the vector to cleave the two reconbinase-recongnizing sequences in the another vector followed with reconstruction to produce a circular DNA molecule formed from a DNA fragment which has existed between the two recombinase-recongnizing sequences and has been cut off from the vector with the recombinase.
- a DNA fragment has an expression unit having a foreign gene and an origin of replication introduced therein
- the DNA fragment autonomously replicates after converted into a circular DNA molecule, and permanently is maintained in the cell to continue to express the foreign gene.
- a recombinant adenoviral vector system is applied to gene therapy, a therapeutic effect is enabled over a long period of time, by a single injection or administration of such vectors.
- an object of the present invention is to provide a recombinant DNA viral vector (1) for transfecting an animal cell, comprising a promoter, a recombinase gene and a poly(A) sequence.
- Another object of the present invention is to provide a recombinant DNA viral vector (2) according to the above vector (1), wherein said DNA viral vector is an adenoviral vector.
- Another object of the present invention is to provide a recombinant DNA viral vector (3) according to the above vector (2), wherein said recombinase gene is recombinase Cre gene derived from E . coli P1 phage.
- another object of the present invention is to provide a recombinant DNA viral vector (4) for transfecting an animal cell, comprising two recombinase-recongnizing sequences, an origin of replication which is operable in the animal cell, a promoter, a foreign gene and a poly(A) sequence, all of said origin of replication, promoter, foreign gene and poly(A) sequence being located between the two recombinase-recongnizing sequences.
- Another object of the present invention is to provide a recombinant DNA viral vector (5) according to the above vector (4), wherein said DNA viral vector is an adenoviral vector.
- Another object of the present invention is to provide a recombinant DNA viral vector (6) according to the vector (5), wherein said origin of replication, promoter, foreign gene and poly(A) sequence are located in this order from the upstream one of the two recombinase-recongnizing sequences.
- Another object of the present invention is to provide a recombinant DNA viral vector (7) according to the above vector (5), wherein said foreign gene, poly(A) sequence, origin of replication, and promoter are located in this order from the upstream one of the two recombinase-recongnizing sequences.
- Another object of the present invention is to provide a recombinant DNA viral vector (8) according to any one of the above vectors (4) through (7), wherein said recombinase-recongnizing sequence is a DNA sequence encoding loxP which is a substrate for recombinase Cre.
- Another object of the present invention is to provide a recombinant DNA viral vector (9) according to any one of the above vectors (4) through (8), wherein said origin of replication is derived from virus or animal cell.
- Another object of the present invention is to provide a recombinant DNA viral vector (10) according to the above vector (9), wherein said origin of replication is selected from the group consisting of origins of replication derived from papovavirus, herpes virus, adenovirus, pox virus and parvovirus.
- Another object of the present invention is to provide a recombinant DNA viral vector (11) according to any one of the above vectors (1) through (10), wherein said promoter and poly(A) sequence are involved in a hybrid promoter (CAG promoter) comprising a cytomegalovirus enhancer, a chicken ⁇ -actin promoter, and a rabbit ⁇ -globin splicing acceptor and poly(A) sequence.
- CAG promoter hybrid promoter comprising a cytomegalovirus enhancer, a chicken ⁇ -actin promoter, and a rabbit ⁇ -globin splicing acceptor and poly(A) sequence.
- another object of the present invention is to provide a method (12) for transducing a foreign gene into an animal cell which comprises the steps of: co-transfecting the animal cell with both a recombinant DNA viral vector comprising a promoter, a recombinase gene and a poly(A) sequence, and a recombinant DNA viral vector comprising two recombinase-recongnizing sequences, an origin of replication which is operable in the animal cell, a promoter, a foreign gene and a poly(A) sequence, all of said origin of replication, promoter, foreign gene and poly(A) sequence being located between the two recombinase-recongnizing sequences; cutting off a DNA fragment containing said origin of replication promoter, foreign gene and poly(A) sequence to produce a circular DNA molecule; and, autonomously replicating said circular DNA molecule within the co-transfected animal cell.
- Another object of the present invention is to provide a method (13) for transducing a foreign gene into an animal cell according to the above method (12), wherein each of said two DNA viral vectors is an adenoviral vector.
- Another object of the present invention is to provide a method for transducing a human gene into a cell which comprises using the above method (12) or (13) in a gene therapy.
- Fig. 1 shows the results obtained by transfecting COS-1 cells or CV-1 cells with combinations of various recombinant adenoviral vectors, recovering DNAs from the transfected cells, digesting the recovered DNA with Hind III, fractionating the DNA fragments by subjecting the treated DNAs to electrophoresis and analyzing by Southern blotting.
- symbols denote as follows:
- the DNA viral vector used in the present invention may be any vectors derived from DNA virus such as an adenovirus that can exist only extrachromosomally after infection. Such DNA virus-derived vectors may be used without any restriction. Examples of such vectors include an adenoviral vector, a vaccinia viral vector and a papovaviral vector.
- adenoviral vector which is a preferred example of the DNA viral vector for transfecting an animal cell and which bears a recombinase gene or a recombinase-recongnizing sequence.
- the adenovirus used in the present invention is an adenovirus which utilizes an animal as a natural host, and a particularly preferred adenovirus is a human adenovirus utilizing a human as a host.
- Human adenoviral genome is a double-stranded linear DNA of about 36 kbp, and has an unique structure in that the DNA strand has an about 100 bp inverted repeat sequence at the both ends and that the DNA strand has further two 55 k proteins which are processed from E2B gene product and which are covalently bound to the 5' end of each of both ends of the DNA strand.
- the genome of the adenovirus used in the present invention is deleted of the E1 region, especially the E1A region. This is because, by being deleted of the E1A region which is associated with a neoplastic transformation activity of adenovirus, the adenovirus is rendered non-virulent and only a foreign gene integrated in the genome is selectively expressed.
- the entire E1A region is not necessarily deleted, but the deletion of the partial E1A region only, especially the 1.3 to 9.3% segment only in the E1A region may attain the desired purpose as stated above.
- the genome in the adenovirus used in the present invention may also be deleted of the E3 region.
- the deletion of 79.6 to 84.8% segment in the E3 region is preferable, because the segment is not essential for replication of the adenovirus.
- the adenovirus used in the present invention is characterized in that the adenovirus cannot propagate in usual host cells, except for a human fetal kidney-derived cell line (293 cell line) wherein the E1A and E1B genes are persistently expressed.
- the recombinant adenoviral vector particles used in the present invention can proliferate in the 293 cell line at a titer level as high as 108 to 109 pfu (plaque forming unit)/ml, same as in wild cell strains.
- the virus particles invade into cells highly efficiently and the virus genome is transferred to the nucleus.
- the adenovirus vector lacks the E1A gene, and native adenoviral promoters in the vector which are activated by the EIA gene product can not become to be operable.
- the foreign gene integrated into the adenoviral genome can be transcribed by the foreign promoter which has been also integrated in the adenoviral genome.
- the recombinant adenovirus particles used in the present invention can minimize adverse affects caused by a native adenoviral genome, and the foreign gene in the recombinant adenovirus vector can be expressed efficiently in various kinds of animal cells.
- the foreign gene in the recombinant adenovirus of the present invention can be expressed in a much wider ranges of cells and tissues. This is because the recombinant adenovirus of the present invention can function efficiently as an expression vector even in a cell wherein an usual adenovirus can not proliferate, as far as the recombinant adenovirus particles can infect and invade into the cell.
- the genome in the recombinant adenovirus of the present invention can not replicate extra-chromosomally and is maintained in the nucleus only for two weeks to two months.
- the repeated administrations of the recombinant adenovirus are required for expressing the foreign gene over a long time of period.
- a problem would be caused that the generation of an antibody may be induced.
- a novel recombinant adenovirus having a recombinase gene is constructed, and on the other hand, another new recombinant adenovirus is also constructed which contains two recombinase-recongnizing sequences which are substrates for the recombinase and which further contains an objective foreign gene and an origin of replication, both of which are located between the two recombinase-recongnizing sequences.
- the two recombinant adenoviruses are co-transfected into an animal cell wherein the recombinase will be expressed. Then, the recombinase acts on the two recombinase-recongnizing sequences to cleave then to form a circular DNA molecule.
- the formed circular DNA molecule containing the origin of replication and foreign gene can autonomously replicate within the co-transfected cells to continue in the expression of the foreign gene.
- the promoters used in the present invention there are an animal viral gene promoter and an animal cellular gene promoter.
- the animal viral gene promoters include a SV40 gene promoter and an adenovirus major late gene promoter.
- the animal cellular gene promoters are a thymidine kinase gene promoter, a metallothionein gene promoter and an immunoglobulin gene promoter.
- a particularly advantageous promoter in the present invention is CAG promoter.
- the CAG promoter is a hybrid promoter comprising a cytomegalovirus enhancer, a chicken ⁇ -actin promoter, and a rabbit ⁇ -globin splicing acceptor and poly(A) sequence.
- the CAG promoter has been reported as a high expression vector in Japanese Patent Application Laid-Open No. 3 (1991)-168087.
- the CAG promoter may be constructed by cutting out it from a plasmid pCAGGS described in the Laid-Open specification supra at page 13, line 20 to page 20, line 14 and page 22, line 1 to page 25, line 6, with restriction enzymes SalI and Hind III.
- the thus constructed CAG promoter may be used in the present invention.
- the recombinase used in the present invention is a specific DNA recombination enzyme, and capable of recognizing a specific DNA sequence to cleave the sequence and exchanging the resulting DNA fragments therewith to relegate those fragments.
- an enzyme there is recombinase Cre encoded by bacterio-phage P1 of E . coli .
- the substrate for this enzyme is a DNA sequence of loxP in bacteriophage P1 [Abremski et al., J. Biol. Chem., 1984, 1509-1514 and Hoess et al., P.N.A.S., 1984, 81, 1026-1029].
- the loxP DNA sequence is a recognition sequence for recombinase Cre.
- Another example of the recombinase is a recombinase encoded by FLP gene derived from yeast 2 ⁇ -plasmid [James R. Broarch et al., Cell, 29 , 227-234].
- a recombinase derived from pSR1 plasmid of Schizosaccharomyces luxii may also be employed. This recombinase is encoded by R gene [Matsuzaki et al., Molecular and Cellular Biology, 8 , 955-962(1988)].
- bacteriophage P1-derived recombinase, recombinase Cre is particularly preferred for the present invention.
- the recombinase Cre gene may be prepared by amplifying the sequence coding the recombinase gene in bacteriophage P1 DNA with polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the other recombinase genes may be prepared with the PCR method in a similar manner.
- Primers used in the PCR method are selected so as to amplify the sequence coding the entire sequence of the recombinase gene.
- the recognition sequence of the recombinase is usually a several tens bp sequence.
- the loxP sequence is composed of 34 bp, and the nucleotide sequences have been identified by Abremski et al., J. Biol. Chem., 1984, 1509-1514 and Hoess et al., P.N.A.S., 1984, 81, 1026-1029.
- the recombinase gene may be chemically synthesized in a conventional manner and provided for use in the present invention.
- the poly(A) sequence used in the present invention is not particularly limited, but a rabbit ⁇ -globin-derived sequence is particularly preferred.
- a nuclear transfer signal sequence together with the recombinase gene into the adenoviral vector.
- the recombinase is transcribed in the nucleus of the cells and then extranuclearly secreted.
- the nuclear transfer signal sequence accelerates the transfer of the recombinase into the nucleus [Daniel Kalderon et al., Cell, 39 , 499-509 (1984)].
- origin of replication used in the present invention which is operable in animal cells
- virus-derived origin of replication examples include those derived from papovavirus, herpes virus, adenovirus, pox virus and parvovirus.
- papovavirus-derived origin of replication there is an origin of replication derived from SV40.
- origin of replication are introduced into the recombinant adenoviral vector of the present invention, whereby the circular DNA molecule cut out by the recombinase can autonomously replicate in the transfected cells.
- the foreign gene used in the present invention are not particularly limited, as far as the gene is expressed under control of the hybrid promoter (CAG promoter) described above or other promoters.
- preferred examples include normal genes which are defective in patients such as adenosine deaminase, dystrophin, low density lipoprotein receptor, ⁇ -1 antitrypsin, blood coagulation factor VIII or blood coagulation factor IX, and galactosidase ⁇ or ⁇ ; cytokines such as interleukins 1 through 12, interferon- ⁇ , ⁇ or ⁇ , tumor necrosis factor- ⁇ or ⁇ , granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin, growth hormone, insulin and insulin-like growth hormone; neurotrophic factors; non-self antigen genes such as allo-HLA (HLA-B7); nucleotide sequences encoding a viral antigen; an antioncogene such as p53, RB,
- the origin of replication, promoter, foreign gene and poly(A) sequence are inserted between the two recombinase-recongnizing sequences in the adenoviral vector, and generally located in this order from the upstream one of the two recombinase-recongnizing sequences.
- the foreign gene, poly(A) sequence, origin of replication and promoter may also be located in this order from the upstream one of the two recombinase-recongnizing sequences. Once they have been included in a circular DNA molecule formed from the adenoviral vector by the recombinase, the above two orders can not be distinguished from each other.
- an animal cell is co-transfected with both the recombinant adenoviral vector expressing the recombinase and the recombinant adenoviral vector bearing the two recombinase-recongnizing sequences and further bearing the promoter, foreign gene and poly(A) sequence, each of which is located between the two recombinase-recongnizing sequences.
- the transfections of the two vectors may be carried out simultaneously or sequentially, because the DNA vectors transferred into the animal cells persist stably over more than one month.
- the recombinant adenoviral vector expressing the recombinase continues to express the recombinase for a certain period of time, whereby the recombinase is continuously produced.
- the produced recombinase acts on the another co-transfected recombinant adenoviral vector bearing the two recombinase-recongnizing sequences to cut out the DNA fragment located between the two recombinase-recongnizing sequences to form a circular DNA molecule.
- the circular DNA molecule has the origin of replication which is operable in animal cells, and therefore autonomously replicates in the co-transfected cells to continue expressing the foreign gene. Accordingly, only the single co-transfection of the two adenoviral vectors can almost permanently continue exhibiting the desired therapeutic effect. It is thus believed that the recombinant adenovirus vectors of the present invention would be extremely effective for gene therapy.
- the gene therapy according to the present invention may be applied in a wide range of human and animal cells such as highly differentiated human and mammal nervous system cells, muscular system cells, hepatic cells, undifferentiated epithelial cells and fibroblast cells.
- adenoviral vector of the present invention It is extremely difficult to construct the recombinant adenoviral vector of the present invention, because the adenoviral genome has proteins covalently linked to the both ends thereof, as described herein-above.
- a process for constructing the another recombinant adenoviral vector bearing two recombinase-recongnizing sequences and further an origin of replication, a promoter, a foreign gene and a poly(A) sequence, each of which is located between the two recombinase-recongnizing sequences is described below in the case of using the origin of replication of SV40.
- the recombinant adenoviral vector of the present invention can be prepared in the same way as the methods in the above 1. (3) to (5)
- the recombinant adenoviral vector bearing the promoter, recombinase gene and poly(A) sequence and the another recombinant adenoviral vector bearing the origin of replication of SV40, foreign gene expression unit and loxP sequence at the both ends may be effectively used for the treatment of various diseases including genetic diseases.
- a high titer viral solution containing the two recombinant adenoviral vectors according to the present invention is appropriately diluted, and the diluted solution may be administered through an appropriate route, e.g., topically (central nervous system, portal vein), orally (using enteric coating), by inhalation, subcutaneously, and the like.
- the mixture was then ligated using T4 DNA ligase (Takara Shuzo Co., Ltd., Japan).
- the reaction mixture was used to transform E . coli JM109 strain (ATCC 53323).
- the treated E . coli cells were inoculated on LB agar plate supplemented with 100 ⁇ g/ml ampicillin, and the transformants growing on the agar were selected to obtain a plasmid pUCCre bearing recombinase Cre gene.
- a cassette cosmid pAdexlcAwt containing CAG promoter which had been prepared according to the method as described in SAIBO KOGAKU (Cell Engineering), 13 , 760-763 (1994), was digested with SwaI.
- the CAG promoter used herein is disclosed as a high expression vector in Japanese Patent Application Laid-Open No. 3 (1991)-168087.
- the CAG promoter may be prepared by excising from a plasmid pCAGGS described in the Laid-Open specification supra at page 13, line 20 to page 20, line 14 and page 22, line 1 to page 25, line 6, with restriction enzymes SalI and Hind III. The thus prepared CAG promoter may be used in the present invention.
- a cosmid may usually efficiently package macromolecular DNA which has been formed by linking with each other in a linear tandem form instead of a cyclic form. 4) After adding 2 ⁇ l of Swal (Boehringer, Germany), the digestion of the cosmid was carried out at 25°C for an hour. The reasons why the cosmid was digested with Swal are given below. If a cassette cosmid is religated without the inclusion of an expression unit therein, a Swal recognition site will be regenerated. Thus, the digestion with Swal can recleave the cosmid having no expression unit included therein, resulting in that no colony is formed. This is a potential method for selecting only a cassette cosmid having an insert sequence.
- the kit can select at a certain extent a cosmid having become a larger size by including an insert sequence. In this experiment, when 10 colonies were picked up, most of them included the insert sequence. Therefore, the clone having the desired orientation (i.e., the left direction which means the direction from E3 gene region to E1 gene region) could be readily obtained.
- the cosmid was manipulated according to a conventional method as described in Izumu Saito et al., JIKKEN IGAKU (Experimental Medicine), vol. 7, 183-187 (1989). 8) The packaged cosmid was infected into E. coli strain DH1 (ATCC 33849).
- the cosmid was inoculated on each of three Ap+ agar plates (supplemented with ampicillin) and 5 ml of Ap+ LB (pool) in amount of each of 1/200, 1/20, 1/2 and the balance, followed with incubation overnight.
- the miniprep DNA from the pool was extracted and prepared.
- a ratio of the cosmid having the insert sequence was examined according to whole enzymatic digestions.
- the colony was picked up together with the agar plate, and cultured in 1.5 ml of Ap+ LB overnight to prepare the miniprep DNA. 9)
- the orientation and structure of the expression unit included in the cosmid were confirmed with digestions with restriction enzymes. That is, a plasmid bearing the expression unit but deleted of most adenovirus DNA was prepared with NruI and ligase, and a DNA fragment was then prepared from the plasmid for final confirmation of cDNA cloning.
- the culture medium containing dead cells was treated aseptically with a sealed type sonicator at the maximum output of 200 w for 2 minutes (30 seconds x 4) to release the virus.
- the precipitates were removed by centrifugation at 3k rpm for 10 minutes at 4°C, and the obtained supernatant was charged at an amount of 2 ml in each of 13 tubes of 5 ml freezing tube.
- the tubes were quickly frozen with dry ice and stored at -80°C. to prepare a third seed solution.
- the third seed solution which contains the recombinant adenoviral vector of the present invention showed a titer as high as 109 PFU/ml.
- the propagated viral DNA was digested with restriction enzymes and then subjected to electro-phoresis.
- the resulting patterns were confirmed by the procedures as described hereinabove. Where there was any doubt that the virus would be possibly mixed with the deleted virus or the parent virus, all of the third seeds were discarded. This is because there would be a possibility that the deleted virus, which had already existed in the second viral solution, rapidly propagated at an appreciable level. Therefore, the above procedures were again performed with another second seed solution.
- the virus solution was purified by subjecting the first seed solutions to a limiting dilution method.
- the recombinant adenoviral vector according to the present invention may be assayed for the titer in a simple manner according to the following procedures.
- each of the recombinant adenoviral vector solution diluted to 10 ⁇ 4 is charged.
- the vector solution is transferred to the wells on the second lane. Thereafter, the same operation is repeated until the 11th lane, and the last 25 ⁇ l of the vector solution is discarded. As the result, the 3 n serial diluted solutions may be prepared until 311 x 10 ⁇ 4. The solution in the 12th lane is non-infected cells as a control.
- adenoviral vector bearing two loxP sequences and further bearing origin of replication of SV40, CAG promoter and hepatitis B virus surface antigen (HBs), each of which is located between the two loxP sequences
- AdexlLCAHBsSL bearing the two loxP sequences and further bearing the origin of replication of SV40, CAG promoter and hepatitis B virus surface antigen (HBs), each of which was located between the two loxP sequences, was obtained by the procedures similar to those of Example 1, (2) and (3).
- COS-1 cells or CV-1 cells were cultured in a 6 cm diameter Petri dish until the cells covered over the entire bottom surface of the dish.
- the recombinant adenoviral vector AdexlLCAHBsSL bears HindIII site of about 6.0 kb and forms a 3.5 kb circular DNA molecule after cleavage with recombinase Cre at the loxP sites. Because this circular DNA molecule has one HindIII site, the recovered DNAs treated with HindIII were analyzed by Southern blotting. A710 bp HBs fragment was used as a probe.
- FCS where it is, e.g., CS
- the cultured cells are washed twice with serum-free medium.
- the viral solution (diluted with serum-free or FCS-supplemented medium) is added during the procedures in such an amount that the cell surface is not dried up.
- the amount is approximately 30 to 40 ⁇ l for a 96-well microplate, 50 to 70 ⁇ l for a 24-well microplate and 100 to 200 ⁇ l for a 10 cm diameter Petri dish. It is practically advantageous to remain intentionally the medium in a small quantity prior to supplementing to the viral solution, then add the viral solution to the remained medium to make the volume as indicated above.
- the viral solution is uniformly spread onto the cells. This operation is carried out 3 times every 20 minutes, and during the operation the cells should be put in a CO2 incubator.
- the time for transfection is generally an hour, and at most, about 2 hours are sufficient for this purpose.
- recombinant DNA viral vectors which may be transfected into a variety of animal cells in such a way that a foreign gene is able to autonomously replicate within the transfected animal cells.
- the present invention also provides a simple process for producing the recombinant DNA viral vectors.
- the recombinant DNA viral vectors of the present invention are useful especially for the treatment of hereditary diseases.
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CN (1) | CN1077919C (fr) |
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AU (1) | AU698250B2 (fr) |
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1996040955A1 (fr) * | 1995-06-07 | 1996-12-19 | Graham Frank L | Vecteurs d'adenovirus pour therapie genique |
WO1997007223A1 (fr) * | 1995-08-18 | 1997-02-27 | Harald Von Melchner | Vecteurs a auto-suppression pour therapie genique |
WO1997009439A1 (fr) * | 1995-09-01 | 1997-03-13 | Genvec, Inc. | Procedes et vecteurs permettant une recombinaison dirigee |
WO1997019181A2 (fr) * | 1995-11-24 | 1997-05-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin | Vecteur de virus utile pour transferer des episomes stables |
WO1997047757A1 (fr) * | 1996-06-12 | 1997-12-18 | Rhone-Poulenc Rorer S.A. | Generation de molecules replicatives in vivo |
WO2000052187A3 (fr) * | 1999-03-05 | 2000-12-21 | Merck & Co Inc | Systeme evolue destine a la creation de vecteurs d'adenovirus |
WO2003002735A2 (fr) * | 2001-06-28 | 2003-01-09 | Phenogene Therapeutiques Inc. | Procedes, vecteurs, lignees cellulaires et trousses permettant de selectionner des acides nucleiques presentant une caracteristique souhaitee |
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- 1995-08-25 AU AU30248/95A patent/AU698250B2/en not_active Expired
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- 1995-08-30 US US08/521,575 patent/US5817492A/en not_active Expired - Lifetime
- 1995-09-13 KR KR1019950029877A patent/KR100379654B1/ko not_active IP Right Cessation
- 1995-09-14 AT AT95306498T patent/ATE321874T1/de not_active IP Right Cessation
- 1995-09-14 DE DE69534902T patent/DE69534902T2/de not_active Expired - Lifetime
- 1995-09-14 EP EP95306498A patent/EP0704534B1/fr not_active Expired - Lifetime
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Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996040955A1 (fr) * | 1995-06-07 | 1996-12-19 | Graham Frank L | Vecteurs d'adenovirus pour therapie genique |
WO1997007223A1 (fr) * | 1995-08-18 | 1997-02-27 | Harald Von Melchner | Vecteurs a auto-suppression pour therapie genique |
US5801030A (en) * | 1995-09-01 | 1998-09-01 | Genvec, Inc. | Methods and vectors for site-specific recombination |
WO1997009439A1 (fr) * | 1995-09-01 | 1997-03-13 | Genvec, Inc. | Procedes et vecteurs permettant une recombinaison dirigee |
US6063627A (en) * | 1995-09-01 | 2000-05-16 | Genvec, Inc. | Methods and vectors for site-specific recombination |
WO1997019181A3 (fr) * | 1995-11-24 | 1997-10-02 | Max Planck Gesellschaft | Vecteur de virus utile pour transferer des episomes stables |
WO1997019181A2 (fr) * | 1995-11-24 | 1997-05-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin | Vecteur de virus utile pour transferer des episomes stables |
FR2749857A1 (fr) * | 1996-06-12 | 1997-12-19 | Centre Nat Rech Scient | Generation de molecules replicatives in vivo |
WO1997047757A1 (fr) * | 1996-06-12 | 1997-12-18 | Rhone-Poulenc Rorer S.A. | Generation de molecules replicatives in vivo |
AU726442B2 (en) * | 1996-06-12 | 2000-11-09 | Gencell S.A. | Generation of replicative molecules in vivo |
US6630322B1 (en) | 1996-06-12 | 2003-10-07 | Gencell Sa | Generating replicating molecules in vivo |
EP1591528A2 (fr) * | 1996-10-18 | 2005-11-02 | CANJI, Inc. | Procédés et compositions pour l'administration et l'expression d'acides nucléiques codant l'interféron alpha |
EP1591528A3 (fr) * | 1996-10-18 | 2005-11-16 | CANJI, Inc. | Procedes et compositions pour l'administration et l'expression d'acides nucleiques codant l'interferon alpha |
WO2000052187A3 (fr) * | 1999-03-05 | 2000-12-21 | Merck & Co Inc | Systeme evolue destine a la creation de vecteurs d'adenovirus |
US8030066B2 (en) | 2000-12-11 | 2011-10-04 | Life Technologies Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
WO2003002735A2 (fr) * | 2001-06-28 | 2003-01-09 | Phenogene Therapeutiques Inc. | Procedes, vecteurs, lignees cellulaires et trousses permettant de selectionner des acides nucleiques presentant une caracteristique souhaitee |
WO2003002735A3 (fr) * | 2001-06-28 | 2003-05-30 | Phenogene Therapeutiques Inc | Procedes, vecteurs, lignees cellulaires et trousses permettant de selectionner des acides nucleiques presentant une caracteristique souhaitee |
WO2003106673A1 (fr) * | 2002-06-17 | 2003-12-24 | 財団法人名古屋産業科学研究所 | Procede d'isolation ou de visualisation selective de cellules cibles differenciees a partir de cellules souches embryonnaires ou kit de visualisation |
Also Published As
Publication number | Publication date |
---|---|
CN1132791A (zh) | 1996-10-09 |
DE69534902T2 (de) | 2006-11-09 |
KR100379654B1 (ko) | 2004-12-03 |
US5817492A (en) | 1998-10-06 |
CA2157063C (fr) | 2005-11-01 |
AU698250B2 (en) | 1998-10-29 |
ATE321874T1 (de) | 2006-04-15 |
NZ272883A (en) | 1996-11-26 |
JP4216350B2 (ja) | 2009-01-28 |
CA2157063A1 (fr) | 1996-03-20 |
AU3024895A (en) | 1996-04-04 |
EP0704534A3 (fr) | 1997-06-11 |
JPH0884589A (ja) | 1996-04-02 |
KR960010864A (ko) | 1996-04-20 |
EP0704534B1 (fr) | 2006-03-29 |
DE69534902D1 (de) | 2006-05-18 |
CN1077919C (zh) | 2002-01-16 |
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